Metarhizium brunneum infection dynamics differ at the cuticle interface of susceptible and tolerant morphs of Galleria mellonella

Abstract

In order for entomopathogenic fungi to colonize an insect host, they must first attach to, and penetrate, the cuticle layers of the integument. Herein, we explored the interactions between the fungal pathogen Metarhizium brunneum ARSEF 4556 and two immunologically distinct morphs, melanic (M) and non-melanic (NM), of the greater wax moth Galleria mellonella. We first interrogated the cuticular compositions of both insect morphs to reveal substantial differences in their physiochemical properties. Enhanced melanin accumulation, fewer hydrocarbons, and higher _L-dihydroxyphenylalanine (DOPA) decarboxylase activity were evident in the cuticle of the M larvae. This "hostile" terrain proved challenging for M. brunneum - reflected in poor conidial attachment and germination, and elevated expression of stress-associated genes (e.g., Hsp30, Hsp70). Lack of adherence to the cuticle impacted negatively on the speed of kill and overall host mortality; a dose of 10⁷ conidia killed ~30% of M larvae over a 12-day period, whereas a 100-fold lower dose (10⁵ conidia) achieved a similar result for NM larvae. Candidate gene expression patterns between the insect morphs indicated that M larvae are primed to "switch-on" immunity-associated genes (e.g., phenoloxidase) within 6-12 h of conidia exposure and can sustain a "defense" response. Critically, M. brunneum responds to the distinct physiochemical cues of both hosts and adjusts the expression of pathogenicity-related genes accordingly (e.g., Pr2, Mad1, Mad2). We reveal previously uncharacterized mechanisms of attack and defence in fungal-insect antibiosis.

Keywords: Galleria mellonella; Innate immunity; cuticle; entomopathogenic fungi; host-pathogen interactions; melanization.

Figure 1.

Integument properties and candidate gene expression of Galleria mellonella morphs. Appearance (a), cuticle thickness (n = 170, *** = p < 0.001), and patterns of melanin deposition (c, d) of melanic and non-melanic morphs. Enhanced expression (mRNA levels) of genes encoding immune factors (gloverin, gallerimycin, apolipophorin III, IMPI, DDC, PPO), stress management (HSP90), detoxification, and cell proliferation of naive melanic (M) larvae compared to non-melanic (NM) larvae (e).

Figure 2.

Biochemical profiles of Galleria mellonella morphs. Quantities and compositions of epicuticular hydrocarbons (a) and fatty acids (b) from un-stimulated (i.e., naive) melanic and non-melanic wax moth larvae. Unpaired t-tests were used to assess differences between each insect morph (* = p < 0.05).

Figure 3.

Development of mycosis (Metarhizium brunneum) in melanic (M) and non-melanic (NM) morphs of Galleria mellonella larvae after topical inoculation. The survival of each insect morph was recorded over 12 days following exposure to two doses of M. brunneum conidia. LС₃₀ values demonstrate similar mortality levels of M and NM larvae in the presence of 10⁷ conidia (p < 0.01, **) and 10⁵ conidia (p < 0.001, ***), respectively (n = 60). The numbers of conidia per 0.25 mm² cuticle were counted on M and NM morphs during infection with M. brunneum (10⁷) (b), and the percentage of germinating conidia (c) that successfully adhered (*** = p < 0.001; n = 22). Penetration of the integument by germinating conidia led to the formation of melanotic lesions (white arrows) on NM larvae – representing de novo synthesis of melanin (d). Conversely, the pattern of melanin deposition on M larvae remained unchanged (e), indicating the melanic-defenses are preformed (images were taken 24 h.p.i. with 10⁷ conidia).

Figure 4.

Gene expression (mRNA levels) of Galleria mellonella integumental tissues exposed to Metarhizium brunneum conidia (LС₃₀) relative to uninfected (control) insects. Insect metalloproteinase inhibitor (IMPI), heat-shock protein (HSP 90), DOPA decarboxylase (DDC), prophenoloxidase (PPO), antifungal peptide gallerimycin, antibacterial peptide gloverin, β-glucan binding protein apolipophorin III, growth factors contig 233 (growth-blocking peptide) and contig 704 (pleiotrophin-like protein). (* = p < 0.05; melanic versus non-melanic at the respective time point).

Figure 5.

Detoxification-associated gene expression in Galleria mellonella morphs exposed to Metarhizium brunneum. Antioxidant enzymes, peroxidase (FPx) (a), glutathione-S-transferase (GST) (b) and transferrin, (c) in the integuments of M and NM larvae at 72 h post fungal infection. The asterisk [*] indicates a significant difference (p < 0.05) between NM (10⁷) and M (10⁵). The hashtag [#] indicates a significant difference (p < 0.01) between NM (10⁷) and NM (10⁵).

Figure 6.

Gene expression (mRNA levels) of Metarhizium brunneum during the infection cycle on melanic and non-melanic morphs of Galleria mellonella. Scheme representing the broad stages of host colonization (a). Subtilisin-like proteases (Pr1a, Pr1b, Pr2), heat-shock proteins (HSP30, HSP70), adhesin-like proteins (MAD1 and MAD2), conidiation – associated gene (cag 8) and nitrogen regulator response (nrr) expression on the insect integuments from 6 to 72-h post-exposure.

Figure 7.

Relative expression patterns of Metarhizium brunneum genes on the integuments of melanic larvae compared directly to non-melanic larvae. Fungal protease Pr2 (a), adhesin-like proteins (MAD1 and MAD2) (b), heat-shock proteins 30/70 (HSP30/70, c/e), conidiation-associated gene (cag 8, d) and nitrogen regulator response (nrr, f).